YhbO protects cells against multiple stresses

Abstract

YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1. A yhbO-disrupted mutant of Escherichia coli is highly sensitive to oxidative, thermal, UV, and pH stresses, and the putative nucleophilic cysteine C104 of YhbO is required for stress resistance. These results suggest that YhbO affects a central process in stress management.

FIG. 1.

Increased sensitivity of the yhbO mutant to environmental stresses. Logarithmic-phase cultures of wild-type (squares) and yhbO-deficient (diamonds) cells were incubated for the indicated times, as described in the text, in the presence of 15 mM (filled symbols) or 50 mM (open symbols) H₂O₂ (A), at 50°C (filled symbols) or 53°C (open symbols) (B), under UV irradiation (C), at pH 2.5 (D), at pH 10.5 (E), or in the presence of 2.5 M NaCl (F), and viable cell counts were determined. Experiments were done three times, and the mean value ± standard error of the mean (SEM) was calculated.

FIG. 2.

Unchanged protein metabolism in the yhbO mutant. (A) Detection of oxidatively modified proteins from E. coli cells after a 50 mM H₂O₂ challenge. Logarithmic-phase cells from the parental strain (lane 1) and from the yhbO mutant (lane 2) were incubated for 40 min at 37°C in the presence of 50 mM H₂O₂, and crude extracts were prepared, electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (10 μg protein in each lane), transferred to a polyvinylidene difluoride membrane, and immunoassayed for protein carbonyls using the OxyBlot kit. (B) 2D gel electrophoresis of insoluble protein fractions after heat shock. The yhbO mutant and its parent were grown at 30°C in Luria-Bertani medium to logarithmic phase and shifted to 53°C for 90 min. Insoluble proteins (15,000 × g pellet) were analyzed by 2D gel electrophoresis, as described in reference , and stained with Coomassie blue (the pH 4 to 7 gradient is from left to right). The amount of protein loaded onto each gel corresponds to identical amounts of bacteria (pellet fractions contain membrane proteins and aggregated protein material [4]).

FIG. 3.

Complementation of the yhbO mutant by its wild-type allele, but not by the C104A mutant. Logarithmic-phase cultures of wild-type cells (filled circles) and yhbO-deficient cells either uncomplemented (empty circles) or complemented with the wild-type yhbO allele (filled squares) or with the C104A yhbO allele (filled triangles) were incubated for 90 min in the presence of the indicated hydrogen peroxide concentration (A) or for the indicated times at 53°C (B), and viable cell counts were determined. Experiments were done three times, and the mean value ± standard error of the mean (SEM) was calculated.